Optimized spirooxindole-pyrazole hybrids targeting the p53-MDM2 interplay induce apoptosis and synergize with doxorubicin in A549 cells

Recently, cancer research protocols have introduced clinical-stage spirooxindole-based MDM2 inhibitors. However, several studies reported tumor resistance to the treatment. This directed efforts to invest in designing various combinatorial libraries of spirooxindoles. Herein, we introduce new series of spirooxindoles via hybridization of the chemically stable core spiro[3H-indole-3,2′-pyrrolidin]-2(1H)-one and the pyrazole motif inspired by lead pyrazole-based p53 activators, the MDM2 inhibitor BI-0252 and promising molecules previously reported by our group. Single crystal X-ray diffraction analysis confirmed the chemical identity of a representative derivative. Fifteen derivatives were screened for cytotoxic activities via MTT assay against a panel of four cancer cell lines expressing wild-type p53 (A2780, A549, HepG2) and mutant p53 (MDA-MB-453). The hits were 8h against A2780 (IC50 = 10.3 µM) and HepG2 (IC50 = 18.6 µM), 8m against A549 (IC50 = 17.7 µM), and 8k against MDA-MB-453 (IC50 = 21.4 µM). Further MTT experiments showed that 8h and 8j potentiated doxorubicin activity and reduced its IC50 by at least 25% in combinations. Western blot analysis demonstrated that 8k and 8m downmodulated MDM2 in A549 cells. Their possible binding mode with MDM2 were simulated by docking analysis.

www.nature.com/scientificreports/ (spectral data provided in ESI; Figs. S1-S41). The absolute configuration of the pharmacophoric spirooxindoles was assigned based on single crystal X-ray diffraction analysis techniques. Based on the X-ray diffraction analysis approved that the reaction is stereoselective and diastereoselective which confirmed the plausible mechanistic pathway via ortho/endo [3 + 2] cycloaddition reaction experimentally favored. The molecular structure of 8p ( Fig. 5; Fig. S42 and molecular packing as indicated in Fig. S43; ESI) and crystal data provided in Tables S1 and S2 (ESI) confirmed the absolute configuration of the desired spirooxindoles.
As for normal non-cancer cells, activated PBL (n = 3) were used and tested against the compounds most active in the two experimental model for the evaluation of antiproliferative activity, that is: 8h, 8m, 8k, and 8j. On average they show a lower antiproliferative activity against activated PBL with a mean IC 50 of 54.7 ± 8.0.
On the whole our compounds 8h, 8m, 8k, and 8j were on average on activated PBL cells about 3.2, 2.7, 2.6, and 2.7 times, respectively, less active than on the tumor cell lines (mean IC 50 values) used as target ( Table 2).
Determination of apoptotic activity. In order to verify the triggering of apoptosis by the administration of our spirooxindole compounds we treated A549 cells with their specific IC 50 values (compounds 8a, 8c, and 8e), and IC 75 s (all compounds), as calculated by the MTT assay. As shown in Fig. 7 and Figure S44 ESI compounds 8t, 8k, and 8m displayed a highly significant apoptotic activity when administered at their specific IC 75 s, as waited for compounds able to inhibit MDM2 protein.
Expression of p53, MDM2, and p21. Among the spirooxindoles under study, those showing the highest apoptotic activity (8k, 8m, 8b) were selected to evaluate their capacity to modulate p53 and its relative targets, namely p21 and MDM2. A549 cells carrying wild type p53 were treated at drug concentrations equivalent to IC 75 ; cisplatin, was also employed as a control of p53 induction. Data on protein expression obtained by western blot (Fig. 8) showed that 8k and 8m did not induce any changes on p53 level but promoted a clear down modulation of MDM2 (all raw materials of immunoblots are provided in Figs. S45-S47 in ESI). On the contrary, the level of p53 significantly increased following 8b exposure and, consequently, an increase of p21 and MDM2 proteins was observed. These data show that in A549 cells, 8b possess some p53 activation capacity comparable to cisplatin; likely the apoptotic activity of 8k and 8m in this cell line is triggered by pathways not involving the activation of p53.
To investigate the effect of other molecules on p53 expression, we performed a dose-response experiment using a concentration range of 0.01, 0.1, 1, 10 µM for each derivative. Cell extracts from 24 h-treated A549 cells were prepared and analyzed by western blot (Fig. 9). In these conditions, 8h, 8j and 8d were able to induce p53 only at high concentrations, while 8k induced MDM2 down regulation at any concentration, confirming the previous observation (Fig. 8).
Next, we evaluated the levels of p53 and its targets in A549 cells treated with low concentration (0.2 µM) of selected derivatives (8h, 8j, 8k and 8d) in combinations with doxorubicin ( Fig. 10) in the conditions used as in Fig. 7. P53 and p21 did not increase in the presence of these compounds alone, while a slight but not significant MDM2 increase was observed, in particular for the 8j molecule. Doxorubicin treatment alone clearly triggered the induction of p53 and p21, but not of MDM2, that instead strongly decreased. When compared to doxorubicin  www.nature.com/scientificreports/ treatment alone, no further increases of p53, MDM2 and p21 proteins was observed in combined treatments (Fig. 10, lower panel). It has to be noted that the combined treatments were conducted at low drug concentrations, according to the experimental conditions used in antiproliferative and apoptotic activity assays. Treatments with a slightly higher drug concentration, but compatible with this experimental setting (0.4 μM), did not show difference. This suggests that likely high concentrations are needed to induce an effect on p53-MDM2 interplay. The abundant increase of p21 after doxorubicin exposure is in keeping with the reported effect of doxorubicin on cell cycle arrest following an activation of p21 by p53 59,60 . However, the 6 h pre-treatment with 8h and 8j before doxorubicin administration, despite increasing the cytotoxicity compared to the single doxorubicin treatment (Fig. 6), did not trigger a further p53 and MDM2 increase, indicating that the MDM2-p53 axis is not involved in the cytotoxicity induced by combination treatment with 8h or 8j and doxorubicin.
Collectively, our data (summarized in Table 3) show that among the spirooxindoles here analyzed, 8h, 8j, 8k, 8m and 8b appear the more promising, although their mode of action needs deeper investigation.
The molecules 8h and 8j which are able to exert antiproliferative activity, as well as to slightly trigger apoptosis, also induce p53, although not at high level and only at high concentrations. The 8b is able to activate p53 and significantly trigger apoptosis. 8k and 8m did not induce p53; instead, they induced a down regulation of MDM2 and a considerable high level of apoptosis.
Thus, these results may suggest that 8h, 8j and 8b have some ability to act on the p53-MDM2 axis although further studies are necessary to define their mode of action. On the other hand, the pathways activated by the other molecules (i.e. 8k and 8m) are likely not strictly related to the inhibition of p53-MDM2 interaction. However, since they showed a high pro-apoptotic activity, may deserve further investigation.
Docking simulations. The X-ray crystal structure of E3 ubiquitin-protein ligase MDM2 co-crystallized with its reference spiro[3H-indole-3,2'-pyrrolidin]-2(1H)-one inhibitor 6SJ was firstly retrieved from the RCSB PDB (PDB ID: 5LAW) 39 then prepared according to the default settings utilizing the "QuickPrep" MOE Version 2016.0802 module 61 . The spirooxindole derivatives modulating MDM2 (8k and 8m) were built in silico and energy minimized, then docked into the inhibitor's binding site as previously reported 44,45 . The docking simulations were conducted employing 'Triangle Matcher' as the placement method and 'London dG' scoring for calculating Gibbs energy for binding. The docking protocol was validated by redocking the co-crystallized inhibitor and reproducing the experimental interactions at acceptable RMSD. Docking results showed that the docked derivatives ( Fig. 11) 8k and 8m accommodated into the MDM2 active site with binding scores (ΔG = −5.94, and −6.01 kcal/mol, respectively) comparable to the redocked reference inhibitor (ΔG = −7.80 kcal/ mol). The best binding mode of 8k displayed its indolinone ring posing π-π interactions with the active site key amino acid residue Lys94 (MDM2) . Nevertheless, the carbonyl linking the heterocyclic core with the installed pyrazole appendage could accept hydrogen bond the same amino acid (Lys94 (MDM2) ). These interactions oriented the molecule so that the installed trifluoromethyl ring was obviously buried in the Phe19 (p53) pocket, the indolinone faced the His96 (MDM2) in the Leu26 (p53) pocket, and the spiro ring was close to Leu54 (MDM2) in the Trp23 (p53) pocket. On the other hand, 8m occupied different spatial orientations, where the indolinone ring fitted into the Trp23 (p53) pocket resembling the reference's core, directing the substituted spiro ring to the Phe19 (p53) pocket and extending the installed substituted pyrazole across the active site towards the His96 (MDM2) in the Leu26 (p53) pocket posing π-π interactions. In light of the results, it is postulated that the synthesized derivatives share some www.nature.com/scientificreports/ key interactions with the reference inhibitor. Installing substituted pyrazole did not hinder accommodation of the molecules into the active site when the spiro-indolinone core is appropriately substituted.

Experimental
Chemistry. The synthesis of chalcones 5a-p required for the synthesis of the desired compounds have been provided in the supporting information.

Conclusion
This study portrays pharmacophoric hybridization design, multicomponent synthesis, molecular modeling studies and biological evaluation of new series of pyrazole-spirooxindoles as direct MDM2 inhibitors. Results demonstrated that the general antiproliferative pattern of the studied 7'-substituted 6'-(pyrazole-4-carbonyl) tetrahydro-3'H-spiro[indoline-3,5'-pyrrolo[1,2-c]thiazol]-2-one echoed the parent spirooxindoline architecture's intrinsic antitumor potential justifying the adopted design rational. The 7'-(2,4-dichlorophenyl) substitution (8h) conferred the highest anticancer potency to the scaffold against A2780 and HepG2 cells, whereas the 3-bromophenyl (8m) and the 4-(trifluoromethyl)phenyl (8k) moieties were the optimized substituents for efficient antiproliferative activities against A549 and MDA-MB-453 cells, respectively. On the other hand, installing pyridin-2-yl (8l) and furan-2yl (8b) on the spiro ring was detrimental to anticancer potencies against all the screened cell lines. Further experiments showed that 8h and 8j potentiated doxorubicin anticancer potential by more than 25% in combinations. Western blot analysis revealed a dose-dependent down regulation of MDM2 in A549 cells after treatment with that 8k and 8m. Docking simulations showed that the promising derivative (8k and 8m) accommodated readily into the MDM2 active site posing various interactions with the key amino acid residues.

Data availability
Data are available from the corresponding authors upon reasonable request.